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Bulletin of the Institute for Chemi University (1964), 42(4): 246-251 Issue Date

1 Studies on the Sulfur-containing Am TitleCompounds in Garlic. (I) : Assimila Garlic Author(s) Sugii, Michiyasu; Suzuki, Tomoji; K Jyoji Citation Bulletin of the Institute for Chemi University (1964), 42(4): Issue Date URL Right Type Departmental Bulletin Paper Textversion publisher Kyoto University

2 Studies on the Sulfur-containing Amino Acids and the Related Compounds in Garlic. (I) Assimilation of Sulfate [35S] in Garlic* Michiyasu Sucii, Tomoji Suzuiu, Toshio KAKIMOTO and Jyoji KATO** (Fujita Laboratory) Received May 19, 1964 For the study of sulfate metabolism during the hydroponic cultivation of garlic plant, tracer technique using 35SO4- was employed. It was found that the roots of garlic possess the high synthetic ability of sulfur containing amino acids and the earliest major labeled amino acids were shown to be cysteine and methionine. After cultivation for 24 hr., the formation of S-allyl-Lcysteine sulfoxide and S-methyl-L-cysteine sulfoxide was observed. In addition to these characteristic sulfur containing amino acids, many unknown sulfur containing amino compounds were detected. INTRODUCTION It is well known that garlic (Allium sativum) contains at least three characteristic sulfur containing amino acids, S-allyl-L-cysteine sulfoxide (alliin),1 S-propyl-Lcysteine sulfoxide2' and S-methyl-L-cysteine sulfoxidc,2) and that these S-alkyl-cysteine sulfoxides are decomposed by the alliinase present in garlic. The biosynthesis of these amino acids is not established yet, and there is no literature concerning the biochemical study on sulfur metabolism in garlic plant. The present paper reports the utilization of inorganic sulfate in growing garlic plants. Using the tracer technique with 35S0,-, the assimilation of 35S in the various periods of cultivation was studied. Material and Method EXPERIMENTAL (1) Cultivation of plants.in April, garlic plants (Allium sativum, white variety) grown-up to about 30cm. in height were lifted from the ground on the 100th day after sowing. After washing with water, the garlic plants were transplanted into hydroponic bottles (one plant per one bottle), and each of which was filled with 100m1. of hydroponic solution. All feedings were made at the same period. The component of the cultivation solution is shown in Table 1. (2) Cultivation of aerial part. The excised aerial part of garlic plant was cultivated with 50 ml. of hydroponic solution in Table 1 containing 950/z C of H235SO4. (3) Incubation of excised roots. Peeled bulbs of garlic were washed with soap and followed by 70% ethanol. After washing with sterile water, they were soaked for * Reported at the 80th Annual Meeting of the Pharmaceutical Society of Japan, Tokyo, April, {- Wi ( 246 )

3 Sulfur-containing Amino Acids and the Related Compounds in Garlic. (I) Table 1. Component of hydroponic solution. Ca (NO3)2 4H200.1 g. KNO g. KH2PO g. MgCl g. FeCI mg. Minor elements*trace H235SO4 (Carrier free)4 inc K2SO40.05 mg. Total 100 ml., ph 5.6 Arnon A5 (B, Mn, Zn, Cu, Mo) and Arnon B5 (V, Cr, Ni, Co, W, Ti)3) were added. 5 min. in a 0.1% HgC12 solution and then washed with sterile water. The sterilized bulbs were placed on the moistened filter paper in a petri dish, and kept at 10-,.15. After 10 days, the growing roots were cut into pieces of about 2 cm. long. They were collected, washed with sterile water, immediately submerged into 20 ml. of a sterile culture solution in Table 2 and were then incubated at Table 2. Component of culture solution.* MgCl mg.MnCI, 4H2O0.09mg. Ca (NO3)2 4H20 4.0mg.ZnC120.03mg. NaCI4.0mg.1-I3B030.03mg. KNO31.6mg.KI0.015mg. KCI1.3mg.Sucrose400.0mg. NaH2P0,, H2O 0.33 mg. Glycine0.06mg. FeC13 6H mg. Nicotinic Acid 0.01 mg. Pyridoxine IIC] 0.002mg. Thiamine HCl0.002mg. H235SO4 (Carrier free) 1 mc Total 20 ml., ph 5.6 "` A modified tissue culture solution of White4) was used. (4) Preparation of protein fraction and amino acid fraction. After a fixed period of cutivation, the roots were washed with water. The plants were rapidly cut into roots, bulbs and aerial part, and to prevent the enzymatic reaction each part was placed in a boiling water for 10 min. and after cooling, the materials were homogenized. The homogenate was adjusted with acetic acid to ph 4.0 and centrifuged to separate insoluble protein fraction. The precipitate fraction obtained was rapidly dried at 100 and stored. To obtain the amino acid fraction, the resulting supernatant solution was passed through a column of Amberlite IR-120 (H-type) and the adsorbed amino compounds were eluted with 40/0 NH4OH and evaporated to dryness under reduced pressure. (5) Analysis of sulfur. Sulfur was estimated as BaSO4 according to the method of Pirie.5) (6) Determination of radioactivity. Radioactivity was determined with a G-M tube. Samples were oxidized to sulfate according to the method of Pixie, adding ( 247 )

4 Michiyasu Sucrr, Tomoji Suzuiu, Toshio KAKIMOTO and Jyoji KA'ro a calculated amount of K2S0, solution as carrier and the sulfate was converted to BaSO, by the usual manner. They awere counted at an infinite thickness and compared with the count of a standard Ba 35S0,. (7) Paper chromatography. Two dimensional ascending paper chromatography was carried out with Toyo No. 50 filter paper 40 x 40 cm. First solvent: PhOH-0.08 % NH2OH (4 : 1), second solvent: BuOH-AcOH-H20 (5 : 1 : 4). Paper electrophoresis was also carried out with Toyo No. 50 filter paper 2 X 40 cm. in pyridine-acetic acid-water buffer (10 : 0.4 : 90, PH 6.5) at 30V/cm. The 35S-labeled. amino acids were detected by radioautography and by color reaction with nihydrin or iodoplatinate reagent. (8) Identification of 35S-labeled amino acids. The radioactive spots on paper chromatogram were identified by cochromatography with the authentic samples. RESULTS AND DISCUSSION (1) Distribution of 35S in each fractions. The distribution of 35S in various fractions after the fixed period of cultivation is shown in Table 3. After 30 min. cultivation, the amino acid fraction of roots showed radioactivity and its specific activity as compared with those of bulbs and aerial part according increased more rapidy with an increment of cultivation time. The specific activity of protein fraction was almost similar to that of amino acid fraction. The specific activity of the amino acid fraction of roots was always higher than those of other organs. However, by additional 5 days cultivation without 3SS0,- after 5 days feeding with 35SOq-, almost no more radioactivity could be found in the amino acid fraction of roots, but the specific activity of the amino acid fraction of bulb increased. From these results it is assumed that during additional 5 days cultivation, 35S-labeled amino acids are transported from roots to bulb. (2) Assimilation of 35S into excised aerial part and roots. Excised aerial part and roots were cultivated in a solution containing 'SW,- and the incorporation of 35S into the amino acid fraction was examined. As shown in Table 4, in aerial part, 15% of 35S in cultivation solution was incorporated into the amino acid fraction, however the specific activity of this fraction was much lower than that of roots. From the results of Tables 3 and 4, it is concluded that the S-containing amino acids synthesized both in aerial part and in roots, and that the roots of garlic possess the high biosynthetic ability of sulfur containing amino acids. For the isolation of 35S-labeled amino acids having high specific activity, the authors used the roots of garlic. Perhaps, sulfur containing amino acids synthetized in both root and aerial part will be transported into bulb, and be stocked there. (3) Identification of 35S-labeled amino acids. 35S-labeled amino acids were identified by the paper chromatography and the autoracliograph.y. The results are given in Fig. 1. After 30 min. cultivation, cystine and three other unknown compounds were detected as 35S-Iabeled compounds. After 2 hr. cultivation, methionine was detected. From these results it is assumed that (cysteine) and methionine are synthesized rapidly from ( 248 )

5 - * Sulfur-containing Amino Acids and the Related Compounds in Garlic. (1) Table 3. Distribution of 35S in amino acid and protein fractions of root, aerial part and bulb of garlic. 30min. 2hr.24hr.10days* Weight12 g Total 35S in extract 12,00 /lc Amino acid Weight75 mg S.5.9 mg Root fraction35s3.16,ttc s.a.**0, Weight0.415g Protein S4.2 mg fraction 35S0.185,tte s.a.** Weight29 g Total 35S in extract7.08 /Lc Amino acid fraction Aerials.a.** Part Weight 142 mg S15.9 mg S0.33i.tc Weight1.92 g Protein S9,8 mg fraction 35S0.078ge s.a.** Weight21 g Total 35S in extract8.33 fec AminoWeight 278 mg acid S24.7 mg Bulb traction35s0.35 ac s.a." Weight1.80 g Protein S5.1 mg fraction 35S0.077itc s.a.** After 5 days' feeding with 35SO 42_, continuously 5 days cultivation without 35SO42-, ** s.a.: specific activity= itc/smg. sulfate in garlic plant. After 24 hr. cultivation, many new radioactive spots were detected. Those spots on the radioautograms of the amino acid fractions of roots were also appeared on the radioautogram of the amino acid fraction of bulb and aerial part. Among the spots, cystine, cysteic acid, methionine, methionine sulfoxide, S-allyl-Lcysteine sulfoxide and S-methyl-L-cysteine sulfoxide could be identified by cochromatography with the authentic samples. After the amino acid fraction was treated with ( 249

6 0 i a Michiyasu SUGII, Tomoji Suzum, Toshio KAKmoTo and Jyoji KATO Table 4. Incorporation of 35S into amino acid fraction of excised aerial part and excised roots. Culture Feeding Weight Total 35S in Amino acid fraction Specific solution times g. extract pc Weight mg. 35S ye pc/mg. Aerial part 95 50miltc. 4 days ml. 1mc3 5in SO 42- Roots44 hr , _..._ 30 min. (root)2 hr. (root)! 1 Cl q ZI: C u)<0i I , - CI) c2, (Z cystine1 1,_, cz,) cystine PhOH-0.08%NH4OH methionine I ' hr. (root) 1:11 0 Q 1 (-0: methionine I I 9 :Viin <::3 <ott,,.., ----n 0 : 10 (---. -) i 4 12 cm... -_--Coysteic cystine S-methylcysteine acid sulfoxide PhOH-0.08%NH4OH b Fig. 1. Radioautogram of 35S-labeled amino compounds in garlic. alliinasel) the spots of the S-allyl-L-cysteine sulfoxide and S-methyl-L-cysteine sulfoxide disappeared. By 24 hr. cultivation, all kinds of the sulfur containing amino compounds in garlic are synthesized and even by additional 10 days cultivation no more S-containing amino acid appeared. The identification of the unknown new amino acids detected on the radioautogram will be reported in the next paper. ( 250 )

7 Sulfur-containing Amino Acids and the Related Compounds in Garlic. (I) REFERENCES (1) A. Stoll and E. Seebeck, Hely. Chinz. Acta, 31, 189 (1948). (2) S. Yurugi, J. Pharm. Soc. Japan, 74, 514 (1954); M. Yoshimura, Vitamins (Kyoto), 14, 627 (1958). (3) D.L. Arnon, Am. J. Bot., 25, 332 (1938). (4) L.L. Reiner and P. R. White, Physiol. Plant., 9, 177 (1956)' (5) N,W. Pirie, Biochem. J., 26, 2041 (1932). (251)

Citation Acta medica Nagasakiensia. 1968, 12

NAOSITE: Nagasaki University's Ac Title Studies on the Unknown Amino Acids Author(s) Yoshikawa, Ichiro Citation Acta medica Nagasakiensia. 1968, 12 Issue Date 1968-03-25 URL http://hdl.handle.net/10069/15532